B cell responses and antibody‐based therapeutic perspectives in human cancers

Abstract Background Immuno‐oncology has been focused on T cell‐centric approaches until the field recently started appreciating the importance of tumor‐reactive antibody production by tumor‐infiltrating plasma B cells, and the necessity of developing novel therapeutic antibodies for the treatment of different cancers. Recent Findings B lymphocytes often infiltrate solid tumors and the extent of B cell infiltration normally correlates with stronger T cell responses while generating humoral responses against malignant progression by producing tumor antigens‐reactive antibodies that bind and coat the tumor cells and promote cytotoxic effector mechanisms, reiterating the fact that the adaptive immune system works by coordinated humoral and cellular immune responses. Isotypes, magnitude, and the effector functions of antibodies produced by the B cells within the tumor environment differ among cancer types. Interestingly, apart from binding with specific tumor antigens, antibodies produced by tumor‐infiltrating B cells could bind to some non‐specific receptors, peculiarly expressed by cancer cells. Antibody‐based immunotherapies have revolutionized the modalities of cancer treatment across the world but are still limited against hematological malignancies and a few types of solid tumor cancers with a restricted number of targets, which necessitates the expansion of the field to have newer effective targeted antibody therapeutics. Conclusion Here, we discuss about recent understanding of the protective spontaneous antitumor humoral responses in human cancers, with an emphasis on the advancement and future perspectives of antibody‐based immunotherapies in cancer.

Recent reports have shown that the main antibody-producing machinery in solid tumors is known as tertiary lymphoid structures (TLS), [22][23][24] which are specialized germinal center-like structures formed by orchestration of conglomerates of B cells and surrounding T cells. 22,25Intratumoral TGF-β silences Satb1, a known genomic organizer, which warrants T follicular helper cell (TFH) differentiation, and adoptive transfer of TFH cells induces intratumoral TLS formation and decreases tumor growth in vivo. 25One area of utmost importance is the effectiveness of these auto-antibodies to govern disease progression.Strikingly, despite extensive spontaneous production of IgG antibodies at tumor beds, the magnitude of the response is not dramatic.In human serous ovarian cancer and endometrial cancer, although IgA is the dominant class of antibody, IgG is also extensively produced. 13,14So far, virtually all approved therapeutic antibody preparations for clinical practice including for cancer treatment are in IgG backbone, 26 while recently the importance of implementing dimeric IgA-based therapeutic antibodies has been underscored. 27,28 this review, we have summarized a comprehensive, up-to-date understanding of how the antibody-producing B cells function within the tumor microenvironment, how the class-switched antibody production is governed and its outcome, and the crosstalk of B lymphocytes with other immune cells in the tumor microenvironment.We have also discussed about major therapeutic antibodies in clinical practice and future prospects of targeting intracellular targets by specialized dimeric IgA antibodies or engineered IgG antibodies.

| FUNCTION OF B CELLS AND PLASMA CELLS IN THE TUMOR MICROENVIRONMENT
Apart from a few instances where research groups have focused on IL-10-producing regulatory B lymphocytes in cancers 29,30 and correlated with immunosuppression, 31,32 growing converging evidence confirms that TIL-B and their antibodyproducing plasma cell subsets predict improved outcomes of patients in multiple cancer types. 7- 9,13,14,33The biological function of B lymphocytes includes antigen presentation to CD4 helper T cells as antigen-presenting cells 34,35 along with their principal function of antibody production when stimulated by cytokines, produced by activated CD4 T cells. 36Besides some virus-induced cancer models, 37 the comprehensive understanding of the antigen presentation function of B cells in solid tumor malignancies is still lacking.In epithelial ovarian cancer, increasing immunogenic advancement of the disease positively correlates with the amount of B cells in the tumor and the extent of antibody production by plasma cells. 38In human endometrial cancer 14 and high-grade serous ovarian cancer, 13 increased infiltration of B lymphocytes has been associated with superior patient survival.Correspondingly, in other epithelial carcinomas, such as in human breast, 10,21 lung, 18 hepatocellular, 5 and colorectal cancers, 3,4 B cell infiltration has been associated with improved response to therapies and better survival.
Equally important, B cell infiltration in non-epithelial cancers such as sarcoma 9 and melanoma 7 is also a predictor of increased survival and therapy responses in patients.Helmink B. and colleagues demonstrated among melanoma patients that, individuals with higher mRNA expression for B cell-and antibody-related genes such as marginal zone b and b1 cell-specific protein, immunoglobulin lambda like polypeptide, or J-chain, in the cancer tissues, positively correlate with response to immune checkpoint blockade therapies and survive more. 7Petitprez F. et al showed that B cells drive immunogenicity in soft tissue sarcoma, and they also demonstrated that B cell signatures in the tumor are associated with better responses to immunotherapies and survival outcomes in patients. 9e main antibody-producing machinery in solid tumors is lymph node-like structures formally known as TLS.0][41] However, the types and extent of different immunoglobulin isotypes produced by TIL-B cells are not consistent across the cancer types, 42 and largely determine the effector functions of spontaneous humoral responses in cancer.Though others have found that plasma cells in the tumor are localized both inside and outside of the TLS, 43,44 and both in tumor islets and stroma, 45,46 Mazor and colleagues interestingly have demonstrated that tumor-reactive IgG antibody-producing plasma cells localize adjacent to the CD20 + B cell cluster in the TLS 34 .1][52][53] The extent of antibody-dependent cytotoxic or phagocytic killing of tumor cells and complement fixation depend on the ratio of the different antibody isotypes produced by the TLSorchestrated TIL-B cells.Therapeutic interventions that enhance the formation and maturation of TLS could boost spontaneous, tumordestroying humoral responses.

| ANTIBODY ISOTYPES, CLASS-SWITCHING, AND THEIR FUNCTION IN SOLID TUMOR CANCERS
Antibodies, in nature, can be one of the five different classes. 54Naïve B cells produce IgM and IgD 55 while antigen experiencing of B cells drives class-switching through V-(D)-J recombination 56 and production of the other three isotypes, IgG, IgA and IgE. 56Immunoglobulin isotype switching from constant-μ (IgM) or constant-δ (IgD) to downstream isotypes constant-α (IgA) or constant-γ (IgG) or constant-ε (IgE) occurs by an event known as class switch recombination (CSR), which is essentially an intra-chromosomal deletional recombination whereas the change from IgM to IgD happens through a process known as alternative splicing of the constant-μ to constant-δ genes. 57,58Each isotype, except IgD, has a unique genomic region, known as the switch (S) region, upstream of each of the constant heavy regions known as the S regions.S regions ($1-12 kb long) are composed of 20-80 bp long, G-rich tandem repeats. 59,60The process of CSR, occurs by end-joining recombination, rather than by homologous recombination, 61,62 involves recombination between the S regions, and results in a change from IgM/IgD on naive B cells to one of the downstream isotypes (IgA/IgG or IgE). 59,60ong the five antibody isotypes, IgG, IgD, and IgE only form monomers while secreted IgA and IgM form dimers and pentamers, respectively. 54The basic structure of all antibodies includes a pair of heavy chain and light chain. 54Though the antigen-specificity of all isotypes of immunoglobulin is determined by the variable region sequences in the heavy and light chains, more precisely, by the complementarity determining regions (CDRs), 54 their function is determined by their Fc regions which also define their isotypes. 54The major effector functions of antibodies are antibody-dependent cellmediated cytotoxicity and phagocytosis (ADCC and ADCP) through the accumulation of cytotoxic NK cells, neutrophils, or macrophages, promoting opsonization and through activation of the complement cascade. 54Different antibody isotypes have different levels of abilities to drive ADCC/ADCP, opsonization or complement fixation. 54e two major isotypes present in human solid tumor cancers are IgG and IgA. 13,14,21,27,63Among the four subclasses of IgG antibodies (IgG1, IgG2, IgG3, and IgG4), IgG1 is the most efficient one for promoting ADCC/ADCP. 64All IgG antibodies fix complement except IgG4, 65 while IgG2 does not function as an opsonin like other IgG subtypes. 65IgA is the most abundant antibody produced in humans, 66 and it is the second most abundant in blood, 66 while more than ninety percent of total IgA belongs to the IgA1 subclass.For polymeric IgA and IgM, the Fc chains of individual monomers are brought together by another small protein known as the J chain. 54A receptor expressed on the basolateral surfaces of mucosal epithelium, known as polymeric immunoglobulin receptor (PIGR) binds with the J chain of polymeric IgA/IgM 54 and governs transcytosis of the antibody 67 where the antibody gets complexed with a fragment of PIGR, known as a secretory component, and released from the apical surface of the mucosal epithelium. 67By this mechanism, polymeric IgA antibodies clear viruses from virally infected cells. 68,691][72] Recent studies suggest that PIGR (and perhaps FCRN) is expressed in several cancer tissues. 73While dimeric IgA binds PIGR expressed on the mucosal epithelium or cancer epithelial cells, 67 monomeric IgA specifically binds to FcαRI (CD89) 74 and Fcα/μR (CD351) 75 while IgG antibodies bind to Fc gamma receptors (FcγRs). 54The significance of aberrant expression of some of these receptors within the tumor microenvironment is still elusive.Theoretically, some of these receptors could sequester tumor-reactive antibodies, produced by TIL-B cells, by re-routing their binding with specific tumor antigens.However, binding of dimeric IgA antibodies with PIGR, which is quasi-universally expressed by virtually all epithelial cancers, elicits transcytosis of dimeric IgA that upregulates DUSP phosphatases, which, in turn, dephosphorylates ERK1/2 and thereby dampens RAS pathway, and also sensitizes the cancer cells for MHCindependent killing by cytotoxic T cells. 13,76IgA-PIGR interaction also thwarts DNA repair pathways and ER stress response proteins such as CHOP. 14Reportedly, CD47-SIRPα checkpoint blockade enhances tumor cell killing by neutrophils, induced by IgA. 77Very recently it has been demonstrated that utilizing the tumor-penetrating abilities of dimeric IgA, intracellular mutated oncodrivers-specific dimeric IgA antibodies could target and expel the oncoproteins out of PIGR + cancer cells. 27The consequence of FCRN expression in cancer is yet to be clearly understood.FCRN is expressed by breast cancer cells which is retained in metastatic cells found in draining lymph nodes, 78 and downregulation of FCRN is associated with aggressive disease progression. 79Similarly, in hepatocellular carcinoma 80 and non-small cell lung cancer, 81 decreased FCRN expression is associated with worse outcome in patients.Interestingly, though NK cells do not express FCRN, fcgrt À/À mice show impaired NK cell development, maturation, and interferon secretion with increased melanoma metastasis into the lung. 82,83Presumably, NK cell development and maturation need neighboring FCRN + immune cells.

| CROSSTALK OF B LYMPHOCYTES WITH OTHER IMMUNE CELLS WITHIN THE TUMOR MICROENVIRONMENT
B cells in the tumor microenvironment do not work alone, rather they work closely with T cells.B lymphocytes and T cells orchestrate to form specialized germinal center-like structures, formally known as TLS.Maturation of TLS into an antibody-producing germinal center is characterized by the presence of high endothelial venules 84,85 and requires expression of BCL6, 86 GL7 25 and cytokine LIGHT. 25In classical TLS, B cells are normally found in the center while T cells are on the periphery (Figure 1A).However, in murine ovarian cancer model, it has been demonstrated that in early developmental stages, B cells rather make the periphery surrounding the central T cell zone. 25The intimate crosstalk between B cells and T lymphocytes in the TLS determines the extent of humoral response within the tumor.The formation of TLS is governed mainly by the T follicular helper (TFH) cells 25,47 and chemokine CXCL13 48 (Figure 1A).While TIL-B cells' activity is positively correlated with effective T cell responses, B cell exhaustion often correlates with the frequency of FOXP3 + regulatory T cells (Tregs). 87Apart from interaction with T cell subsets, much is not known yet about the interactions of TIL-B cells with other immune cells in the tumor microenvironment.Among other cells in the tumor microenvironment, NK cells could promote B cell activation and antibody class-switching by CD40-CD40L interaction and IFN-γ production 88,89 (Figure 1B).A major proportion of human γδ cells, (Vγ9Vδ2 cells) express CXCR5 and therefore could co-infiltrate the tumor microenvironment with CXCR5 + B cells 90 (Figure 1A).Similar to NK cells, γδ cells also promote class-switched antibody production 90,91 (Figure 1B).Nonetheless, tumor-associated macrophages (TAMs) secrete B cells-activating factor (BAFF) and a proliferationinducing ligand (APRIL) and govern B cell proliferation, 92 and also support B cell development by secreting IL-6 93 (Figure 1B).Chen Z et al found that TAMs drive IgG-producing plasma cells in hepatocellular carcinoma. 63In a very intriguing study, Shaul M et al have demonstrated that the differentiation of tumor-infiltrating CD45 + B220 + CD138 À B cells into IgG-producing B220 + CD138 + plasma cells requires physical contact with tumor-associated neutrophils, which they found to be independent of the involvement of tumor-infiltrating T cells. 94Notably, they have also found that tumorassociated neutrophils drive B cell recruitment to the tumor microenvironment by secreting TNF-α. 94

| THERAPEUTIC ANTIBODIES IN THE CLINICS AND FUTURE PERSPECTIVES
Despite the huge early success of two monoclonal antibody-based immunotherapies for cancer, rituximab 100,101 and trastuzumab 102,103 against CD20-positive B cell lymphoma and Her2 positive estrogen receptor (ER)/progesterone receptor (PR) negative breast cancer, respectively, the field of immunotherapy research gradually became more focused on cellular therapies such as adoptive TIL therapy, 104,105 CAR T cells therapy, 106,107 CAR NK therapy 108,109 etc. after their success in solid tumors.The importance and effectiveness of these cellular therapies are unquestionable, however, the cost of such treatments is very expensive in their current states 110 while antibody-based therapies are significantly cost-effective and therefore affordable to a larger proportion of eligible patients.Primarily the effector functions of immunotherapeutic antibodies against cancer lie in their ability to induce ADCC/ADCP or complement fixation.Rituximab, a monoclonal IgG1 antibody, binds with CD20 on malignant B cells and induces NK cells-and macrophage-mediated killing or complement-mediated lysis of the cancer cells. 111Similarly, Daratumumab, a monoclonal IgG1 antibody, binds with CD38 on myeloma cells and is extensively used for treating multiple myeloma patients. 112Resistance to rituximab in relapsed refractory lymphoma patients is common 113 and therefore alternative approaches have been introduced later in the clinical practice, for example targeting CD79b by Polatuzumab 114 or using bispecific antibodies, such as Glofitamab which is a CD20-directed, CD3 T cells engager. 115,116Trastuzumab, which is also an IgG1 monoclonal antibody, works in a similar fashion to Rituximab or Daratumumab by binding with surface protein Her2 on breast cancer cells and is a life-saving drug for ER/PR negative Her2 positive breast cancer patients. 117Apart from killing target cancer cells, rescuing antitumor cytotoxic functions of TILs resulted in significant increase in antitumor immunities and corresponding reduction in cancer progression by targeting CTLA-4 118,119 and PD-1 120,121 by monoclonal antibodies were demonstrated in parallel by James P. Allison and colleagues; and Tasuku Honjo and associates, respectively, popularly known as immune checkpoint blockade (ICB) therapies, which produced the Joint-Nobel prize in physiology or medicine in the year 2018. 122The anti-PD-1 antibodies (Nivolumab or Pembrolizumab) are made in IgG4 backbone, 123,124 to annihilate the elimination of antibody-bound PD-1 positive T cells by ADCC/ADCP or complement, though the anti-CTLA-4 antibody, Ipilimumab, was developed in human IgG1 backbone. 1257][128][129] Approaches for targeting intracellular oncoproteins and mutated oncodrivers have been restricted to the development of novel small molecule inhibitors. 130Some of them are like different EGFR inhibitors. 131recently FDA-approved Sotorasib 132 targeting G12C-mutated KRAS or MRTX1133 133 targeting KRAS G12D which is awaiting FDA-approval.
Unfortunately, small-molecule inhibitors have a smaller half-life than antibodies 134 and the development of resistance is very common. 135,136Dimeric IgA antibodies, which bind naturally expressed PIGR 137 on the basolateral surface of mucosal epithelium, through their J-chain, 54 and undergo transcytosis by taking a portion of PIGR (known as secretory component), and released by the apical surface, 67 and through this mechanism they clear viruses from the gut. 68,69tably, PIGR is heavily expressed in a majority of the epithelial cancer types and it has been exhibited that dimeric IgA can also undergo PIGR-driven transcytosis through cancer epithelial cells, 13,14 which instigated their use for targeting intracellular oncoproteins.Recent findings demonstrated that specific dimeric IgA antibodies, but not the same antibodies in IgG backbone, against G12D mutation of KRAS and R132H mutation of IDH1, could target KRAS G12D and IDH1 R132H , respectively, inside the cancer cells harboring these mutations and significantly abrogate tumor progression in vivo 27,138 (Figure 2).This opens a novel opportunity to develop dimeric IgA-based (or perhaps engineered IgG with the ability to bind and internalize with PIGR) therapeutic antibodies against virtually any intracellular oncoproteins.
In future, strategies such as developing antibodies permeable through the cell membrane and reachable to the subcellular locations like the nucleus (for example against mutant p53) could shift the paradigm of antibody-bases immunotherapies against cancer and other diseases.is also important, pertinent to the fact that IgG antibodies have a much higher half-life, and therefore industrial production of monomeric IgG is cost-effective than dimeric IgA antibodies.
In a mouse model of lung cancer, Wang Y et al demonstrated that with an increasing number of myeloid-derived suppressor cells (MDSCs)-infiltration into the tumor mass they observed impaired B cell functions, indicated by decreased serum IgG levels. 95MDSCs 96 in the tumor microenvironment force B cell differentiation into IL-10, IL-35, and TGF-β-producing 97 immunosuppressive regulatory B cells (Bregs), 98,99 however, Bregs do not have any unique identification marker like FOXP3 for Tregs, and therefore the classification of Bregs among total B cells is elusive.

1
Tumor microenvironment and B lymphocytes.(A) CXCL13, secreted in the tumor microenvironment, attracts CXCR5 + B cells, CXCR5 + TFH cells, and CXCR5 + Vγ9Vδ2 γδ T cells that together promote formation of tertiary lymphoid structures.(B) NK cells promote B cell activation by engaging with CD40 on B cell surface through CD40L.NK cells and γδ T cells promote B cell classswitching by secreting IFN-γ.Tumorassociated macrophages promote B cell development, proliferation, and survival by secreting B cells-activating factor (BAFF) and a proliferation-inducing ligand (APRIL).

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| CONCLUDING REMARKS Though the role of T cells in antitumor immunity is indisputable, it is time to appreciate the importance of coordinated humoral and immune response in human malignancies and look for strategies that induce sustained, robust antitumor immune responses by driving simultaneously both the arms of immune system.Apart from the suitability of targeting intracellular targets by dimeric IgA antibodies, their suitability to synergize with existing immunotherapies and chemotherapies should also be evaluated.Previously it has been shown that non-antigen specific dimeric IgA could enhance T cell-mediated killing of PIGR-expressing cancer cells in an MHC-I independent manner, therefore, there is a high chance that therapeutic use of dimeric IgA antibodies could also promote cytotoxic killing by CD8 T cells, in addition to ADCC/ADCP.The development of penetrable IgG antibodies F I G U R E 2 Intracellular targeting of mutated oncoproteins by dimeric IgA antibodies.Mutation-specific dimeric IgA antibodies can bind with PIGR expressed on the epithelial cancer cells of major cancer types and get internalized with a fragment of PIGR known as the secretory component.Internalized dimeric IgA antibodies encounter target antigens in the cytoplasm and could haul them outside of the cells through transcytosis whereby the mutated oncodrivers could be expelled into the extracellular space by secretory IgA.Treatment of PIGR + tumor cells sensitizes them for CD8 + cytotoxic T cell-mediated killing.